During the past 25-30 years the two grants under consideration for merger in the MIRA program (GM46638 and GM34431) have provided numerous insights into the mechanisms underlying the control of gene expression during animal development. Specific highlights include evidence that Hox proteins function as sequence-specific transcription factors, the characterization of the complex eve stripe 2 enhancer, and the elucidation of the affinity threshold model for the differential regulation of gene expression by te Dorsal (NF-kB) gradient. These studies have established the early Drosophila embryo as a premiere system for the study of gene activity in animal development. It is the goal of the proposed MIRA grant to exploit the advent of new imaging technologies to uncover novel mechanisms of gene control. During the past year we have begun to use live imaging methods to visualize the dynamic regulation of gene expression during Drosophila embryogenesis. These studies have led to a number of striking observations that we wish to follow-up during the upcoming funding period, including transcriptional bursts of eve stripe 2 expressions, the non-additive activities of shadow enhancers, transcriptional memory, and allelic communication. We will determine whether transcriptional bursting is due to unstable enhancer-promoter looping interactions, and examine the possibility that the two alleles at a given genetic locus somehow communicate to ensure balanced levels of expression during development. The research plan includes four specific aims: 1) investigate the dynamics by which a single enhancer activates two different target promoters; 2) explore the possibility that temporal precision depends on enhancer switching, whereby enhancers with overlapping activities work in a sequential manner during development; 3) determine whether transcriptional memory depends on specific histone modifications such as methylation; and 4) substantiate our preliminary evidence for allele communication by examining a variety of sensitized transgenes in living embryos.